1. Field of the Invention
The invention relates to the in vitro multiplication of plants.
2. Discussion of the Background
Plants which are multiplied in vitro tend to display growth disturbances which disrupt the industrial production of these plants by micropropagation. The affected explants (offshoots) bear abnormal leaves that are more or less rolled up, translucid, often displaying a dark green color. The explants lose their capacity to proliferate, they canker and die more or less rapidly.
This problem is most often called "vitrification", "vitrescence" or "hyperhydria", and it will be referred to hereafter as "vitrification". This phenomena is all the more frequent or severe when the plants are subjected to intense multiplication.
The use of liquid media also increases the risks of vitrification. NAVATEL (Fruits, vol. 37, n.degree. 5, pp. 331-336, 1982) described the problem as follows: "This symptom which generally appears during the multiplication phase has been noted by many laboratories. The microcuttings or microplants adopt a "vitreous" aspect. The leaves which are dark green and shiny, bend, and become brittle and translucid. The significance of the damage appears to increase with the number of cultures grown on the proliferation medium which has a high cytokinine content. In most cases, the seedlings which display those symptoms become stymied and they are nearly no longer usable for subsequent multiplication. Sometimes we observe a certain restoration of the plant when it is placed in a medium free of auxine and cytokinine. This kind of phenomena has been observed on the GF 677 and also on various stocks from cherry-trees and appletrees.
"Many hypotheses have been set forth to try to explain the origin of these symptoms; too rich a medium, toxicity of the NH.sub.4.sup.+ ion, excessive density inside the jar, ethylene production, ill adaptation of the culture jars, toxicity by way of accumulation of specific growth substances (cytokinine) inside the plant. Recent work indicate that the appearance of that kind of symptom might be connected with the matrix potential of the medium (DEBERGH et al, 1981). Other authors confirm the predominant role that the NH.sub.4.sup.+ ion allegedly plays, perhaps in correlation with other elements, in the appearance of that phenomenon (BEAUCHESNE, 1981).
Other observations have been made by different authors concerning techniques that make it possible to eliminate or at least to reduce the severity of those symptoms. ZUCCHERELLI (1979) points out that the addition of food pectin to the culture medium might reduce the occurrence of "vitereous" plants. DRUART (1980) indicates that the passage of plant material into a cold chamber at more or less 2.degree. C. for about one month allegedly restricts the appearance of such symptoms.
"However, those findings do not make it possible to explain wholly this phenomenon, which is probably very complex as a result of the number of factors that can be involved."
Agar is presently used in culture media, usually in amounts of 7 to 12 g/l, in order to solidify the media. This facilitates the arrangement of plants and reduces to a certain extent the risks of vitrification. However, the rate of multiplication is always higher in a liquid medium than it is in a solid medium, especially when the amount of agar is greater than about 7 g/l. The solutions recommended thus far have only brought a very limited solution to vitrification, with the symptoms re-emerging after 2 or 3 multiplications (generations) on the recommended improved medium.
With respect to DEBERGH's (Physiol. Plant 59.270-276, 1983) recommendation to increase the amount of agar in the solid medium, this approach can protect microcuttings or microplants from vitrification, but it leads to a very high reduction of the rate of multiplication. The effect of agar in this instance is simply to increase the matrix potential of the medium.
This approach is very different from the approach used in the present invention in which a liquid medium is used. The present invention uses a null matrix potential. Furthermore, increasing the amount of agar augments the solid feature of the culture medium, and reduces contact between the microcuttings or microplants and the culture medium. This diminishes even more the rate of multiplication. The relative "protection" against vitrification is obtained to the considerable detriment of multiplication rate.
There is therefore a strongly felt need for a solution to the problem of "vitrification", "vitrescence" or "hyperhydria" found in the in vitro multiplication of plants, where such a solution does not act to the detriment of the multiplication rate of the plants being grown.